Apparatus and Method for Differentiating Multiple Fluorescence Signals by Excitation Wavelength

ABSTRACT

An apparatus and method are provided for differentiating multiple detectable signals by excitation wavelength. The apparatus can include a light source that can emit respective excitation light wavelengths or wavelength ranges towards a sample in a sample retaining region, for example, in a well. The sample can contain two or more detectable markers, for example, fluorescent dyes, each of which can be capable of generating increased detectable emissions when excited in the presence of a target component. The detectable markers can have excitation wavelength ranges and/or emission wavelength ranges that overlap with the ranges of the other detectable markers. A detector can be arranged for detecting an emission wavelength or wavelength range emitted from a first marker within the overlapping wavelength range of at least one of the other markers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/642,009 filed Dec. 18, 2009, which is a continuation of U.S.patent application Ser. No. 10/440,852 filed May 19, 2003, now U.S. Pat.No. 7,735,588, which claims benefit under 35 U.S.C. §119(e) fromProvisional Patent Application No. 60/381,671 filed May 17, 2002,Provisional Patent Application No. 60/409,152 filed Sep. 9, 2002, andProvisional Patent Application No. 60/450,734 filed Feb. 28, 2003. Allpatents, patent applications, and publications mentioned herein areincorporated herein in their entireties by reference.

BACKGROUND

For identification of biological and chemical sample compositions,various quenched or unquenched dyes can be added to a sample. Anunquenched dye fluoresces at a particular emission wavelength whenexposed to excitation light. Quenching dyes can greatly reduces theamount of light emitted by that dye at particular wavelengths.

The apparatus and methods currently in use for sample componentdetection are expensive, and can require a broad spectrum of light,multiple light sources, and/or multiple detectors, for recordation andinterpretation of particular emitted wavelengths from a given sample.For example, in the past, the presence of the components in a sample hasbeen determined by detecting and differentiating specific emissionwavelengths emitted from a sample, requiring relatively expensivedetectors for detecting and differentiating emission wavelengths. It isdesirable to create a less expensive apparatus and method of determiningthe composition of a sample using fluorescent dyes.

SUMMARY

An apparatus and method for differentiating multiple fluorescencesignals by excitation wavelength are set forth. The apparatus and methodcan allow for the determination of a sample composition using specificexcitation wavelengths or wavelength ranges, a plurality of detectablemarkers, for example, fluorescent dyes, and one or more detectors. Theadvent of inexpensive light sources, such as, for example, lightemitting diodes (LEDs), allows the apparatus and method of the presentteaching to provide multiple excitation wavelengths or wavelength rangesat a low cost, and to economically and efficiently determine thepresence or absence of target components of a sample composition.

According to various embodiments, the apparatus can include a samplewell and a sample arranged in the sample well. The sample can include aplurality of components including a plurality of fluorescent dyes addedthereto. Each of the plurality of fluorescent dyes can exhibit anexcitation wavelength range and an emission wavelength range. Theemission wavelength range can overlap with an emission wavelength rangeof one or more of the other fluorescent dyes.

According to various embodiments, an optical instrument can be providedthat includes a light source arranged to emit an excitation wavelengthor wavelength range toward a region capable of retaining a sample, suchthat a fluorescent dye, if present in the region, can be caused tofluoresce. The light source can provide excitation wavelength rangesthat correspond to respective excitation wavelength ranges of aplurality of fluorescent dyes. A detector capable of detecting anemission wavelength emitted from a fluorescing dye can be used todetermine the absence or presence of a component associated with thedye. For example, the dyes can include intercalating dyes, reporterdyes, free-floating dyes, and the like.

According to various embodiments, the light source of the apparatus canbe an array of light generating sources, such as an array of LEDs,lasers, or both, for example. The apparatus can further include anexcitation wavelength-excluding device, an excitation wavelengthfocusing device, or both.

According to various embodiments, one or more of the light source andthe detector can be separated from the sample by a lightpipe.

According to various embodiments, a method of differentiating multiplefluorescent signals by excitation wavelength can be provided. The methodcan include providing a sample including a plurality of components in asample well and adding a plurality of fluorescent dyes to the sample.Each of the plurality of fluorescent dyes can possess a respectiveexcitation wavelength or wavelength range and an emission wavelength orwavelength range. A respective excitation wavelength range can bedirected from a light source toward the sample to excite a fluorescentdye when in the presence of a target component. Emission wavelengthsthat are emitted from the fluorescent dye in the sample can be detected.The presence of a particular component in the sample can be determinedby the detection of a corresponding emission wavelength. The method canthen be repeated utilizing different excitation wavelengths to determinethe presence of other particular components in the sample. Becauseexcitation spectra are broad, a single excitation wavelength can excitemultiple dyes simultaneously. Excitation wavelengths can be chosen so asexcite a first dye more strongly compared to other dyes. The other dyescan be excited weakly compared to the first dye, or not at all in thepresence of the respective excitation wavelength or wavelength range.The response of each possible dye versus excitation wavelength can bemeasured. The response of a sample of unknown constituents can bemeasured. Linear algebra techniques can be used to solve a system oflinear equations. The solution set thus generated can describe theconcentrations of the dyes in the sample of formerly unknownconstituents.

According to various embodiments, a method of differentiating multiplefluorescent signals by excitation wavelength can include multiple lightsources forming excitation beams in various excitation bands. The methodcan include a multi-notch filter. A plurality of overlapping multi-notchfilters can be utilized in the method with at least one multi-notchfilter being used as an excitation filter and at least one multi-notchfilter being used as an emission filter.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the present teachings are exemplified in theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and known to those of ordinary skill in theart. In the drawings:

FIG. 1 depicts a side view of various embodiments;

FIG. 2 is a graph showing the excitation and emission frequencies of twodyes, and the frequency of a bandpass filter for use in variousembodiments;

FIG. 3 is a table showing the relative amount of excitation of two dyesat two separate wavelength ranges;

FIG. 4 depicts an embodiment including a light source directed through aprism;

FIG. 5 depicts another embodiment of the present teachings;

FIGS. 6 a-c depict several elements of various embodiments of theteachings, wherein FIG. 6 a is a side view of a single sample well witha removed light source, FIG. 6 b is a top view of a multiple sample wellarray depicting a light source and detection array, and FIG. 6 c is atop view of an array of excitation wavelength excluding devices usedwith the light source and detection array of FIG. 6 b;

FIG. 7 depicts various embodiments having one light source emittingexcitation wavelengths toward multiple sample wells, each well having arespective detector; and

FIG. 8 depicts various embodiments wherein a light source, multipledetectors for each sample well, and a grating is used to separate theemission beam according to various wavelengths per grating diffraction;

FIG. 9 illustrates an exemplary embodiment of a light source layout, forexample, an organic light emitting diode (OLED) layout; and

FIG. 10 illustrates an exemplary embodiment of a light source layout,for example, an OLED layout with varying color OLEDs stacked upon eachother.

FIG. 11 is a schematic diagram of an optical instrument and an opticalpathway generated by the instrument according to various embodiments.

FIG. 12 is a schematic diagram of an optical instrument that includes aplurality of LEDs, a plurality of collimating lenses, a plurality offilters, a plurality of reaction region lenses, a plurality ofspaced-apart reaction regions, and a detector, and the optical pathwaygenerated by the optical instrument, according to various embodiments;

FIG. 13 is a perspective view of an optical instrument that includes aplurality of LEDs, a plurality of collimating lenses, a plurality offilters, a plurality of reaction region lenses, a plurality ofspaced-apart reaction regions, and a detector, and the optical pathwaygenerated by the optical instrument, according to various embodiments;

FIG. 14 is a schematic view of an optical instrument and an opticalpathway generated by the optical instrument.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the variousembodiments of the present teachings.

DESCRIPTION

According to various embodiments, for example, the embodiment shown inFIG. 1, an apparatus can include at least one sample well 10 adapted toreceive a sample 12 containing at least two fluorescent dyes, a lens 14,a light source 16 capable of emitting at least two excitationwavelengths, and a detector 18 for detecting emission wavelengths.According to various embodiments, the apparatus can also include one ormore excitation wavelength-excluding devices 22, one or more excitationwavelength-focusing devices, one or more lightpipes 24, one of morelenses 14, or combinations thereof.

According to various embodiments, the sample well 10 can be adapted toreceive a sample 12 containing two or more components to be identified.For example, the sample 12 can be biological or chemical substances insolution, such as a sample containing DNA sequences for nucleic acidsequence amplification and/or sequencing detection. The sample 12 can beinserted into the sample well 10 directly, or the sample well 10 can beadapted to receive a sample container, for example, a cuvette or testtube. The sample well 10 can be a single sample well, or an array ofsample wells arranged in a block, such as, for example, an array of 96sample wells. Each sample well 10 can be heated and/or cooled duringoperation. Alternatively, the block can be capable of heating andcooling each sample well situated therein.

FIG. 11 shows an exemplary instrument according to various embodimentsthat can include a reaction region holding assembly 1148, for example, athermal cycler block, including wells 1144 for holding respectivereaction regions 1140, for example, vials, spaced apart from oneanother. The reaction regions contain respective samples 1142. Thesamples can be, for example, respective suspensions of ingredients forpolymerase chain reaction (PCR). If the reaction region holding assembly1148 is a thermal cycler block, the assembly 1148 can include a thermalcycle controller 1149 for cycling the temperature of the block through atemperature program.

Each reaction region 1140 can include, for example, any chamber, vessel,container, sample well, capsule, vial, sample array, centrifuge tube, orother containing, restraining, retaining, or confining device, withoutlimitation, that is capable of retaining one or more samples forfluorometric analysis or illumination thereof. The reaction regions 1140can be fixed, secured, mounted, or otherwise attached or connected to,separate from, or integral with, the reaction region holding assembly1148. The assembly 1148 can be attached or connected to, or placed on, asurface of a substrate or a holder and positioned to enable one or moreof the reaction regions to be illuminated by a light source. The holdingassembly can be, for example, a purification tray, microtiter tray,multiwell tray, sample array, micro-well array or like device forholding multiple samples.

The samples 1142 to be analyzed can include aqueous suspensions ofsample materials, for example, that might include a “seed” sample of atarget nucleic acid sequence, selected primers, nucleic acids, enzymes,buffers, and other chemicals conventionally used for PCR, for anisothermal reaction or another DNA amplification method well known inthe art.

The reaction regions 1140 can be heated and cooled in a predeterminedcycle by electric heaters, liquid or air coolants, or a combination ofthese, or by other methods to achieve thermal cycling. The reactionregions 1140 can be cycled between two temperature phases so as toaffect PCR, for example.

According to various embodiments, two or more components of the sample12 can be identified by adding a number of fluorescent dyes to thesample 12. Each component can be positively identified by thefluorescence of a dye.

According to various embodiments, each fluorescent dye can be selectedso as to become unquenched due to the presence of a particular targetcomponent in the sample. Each fluorescent dye can be selected to possessparticular excitation and emission wavelength ranges. After the dye hasbeen added to the sample 12, a wavelength of light corresponding to theexcitation wavelength range of the dye can be directed at the sample 12by the light source 16, causing the dye to fluoresce when in thepresence of a target component. The fluorescing dye can emit light at anemission wavelength that can be detected by a detector 18. Accordingly,the presence of the particular component in the sample 12 can bedetermined by detecting whether an emission beam at a particularwavelength or wavelength range is emitted from the sample 12 when acorresponding excitation beam is directed to the sample.

According to various embodiments, the presence of various dyes in asample utilizing respective excitation wavelength ranges can be used toidentify the various components. The plurality of fluorescent dyes canbe chosen such that each dye possesses (a) a discrete or substantiallydiscrete excitation wavelength range, and (b) an emission wavelengthrange that overlaps with an emission wavelength range of one or more ofthe other fluorescent dyes. For example, the excitation and emissionwavelengths of exemplary dyes 5-FAM™ and TET™ are depicted in FIGS. 2and 3. As shown, 5-FAM™ and TET™ substantially discrete excitationwavelength ranges centered at 470 nm and 525 nm, respectively. Moreover,referring to FIG. 2, within the wavelength range of approximately >600nm, 5-FAM™ and TET™ possess substantially overlapping emissionwavelengths. FIG. 2 depicts the emissions of the dyes using thefollowing representation: TET Ex as a solid-line with x's through theline; 5-FAM Ex as a dashed-line using small dashes; TET Em as adashed-line using long dashes; 5-FAM Em as a solid-line; and emissionsafter bandpass filter processing with o's through the line.

According to various embodiments, PCR dyes can be used that onlyfluoresce when bound to a target molecule. Nucleic acid sequenceamplification dyes can also be attached to probes that also areconnected to quenchers, and the action of nucleic acid sequenceamplification enzymes will disassemble the dye-probe-quencher moleculecausing the dye to increase its fluorescence. Nucleic acid sequenceamplification can be performed using a variety of methods, for example,polymerase chain reaction (PCR), isothermal amplification reaction, wellknown in the art. When a PCR procedure is used, for example, the numberof unquenched dye molecules doubles with every thermal cycle.Fluorescing dyes are well known in the art, and any of a plurality offluorescent dyes having various excitation wavelengths can be used.Examples of such dyes include, but are not limited to, Rhodamine,Fluoroscein, dye derivatives of Rhodamine, dye derivatives ofFluoroscein, 5-FAM™, 6-carboxyfluorescein (6-FAM™), VIC™,hexachloro-fluorescein (HEX™), tetrachloro-fluorescein (TET™), ROX™, andTAMRA™. Dyes or other identifiers that can be used include, but are notlimited to, fluorophores and phosphorescent dyes. Dyes can be used incombinations of two, three, four, or more dyes per sample. According tovarious embodiments, the family of 5-FAM™ 6-FAM™, VIC™, TET™, and/orROX™ dyes can be used to indicate the presence of sample components.

According to various embodiments, there are a number of dyes that can beused in nucleic acid sequence amplification detection. The detection canbe real-time. Some dyes weave themselves into the DNA and increase theirfluorescence once they do. For these intercalating dyes, thefluorescence strength grows as the number of DNA molecules grows. Thisintercalation does not have to be sequence specific. Samples can bedifferentiated by looking at the DNA melting temperature. More commonly,a dye can be attached to one end of a probe DNA sequence and a quenchercan be attached to the other end. When the probe binds to a targetsequence, an enzyme can cleave the probe into pieces, unbinding thequencher and dye so the dyes can be excited and can release their energyvia fluorescence rather than having it absorbed by the quencher.According to various embodiments, free-floating dyes can be used.

According to various embodiments, various detectable markers can beused, in addition or in alternate, to dyes. Markers can include, forexample, fluorescing dyes, free-floating dyes, reporter dyes, probedyes, intercalating dyes, and molecular beacons. Dyes that fluorescewhen integrated into DNA can be intercalating dyes. Other dyes known as“reporter” dyes can attached to the ends of “probes” that have“quenchers” on the other end. A nucleic acid sequence amplificationreaction, for example, PCR, can result in the disassembly of theDye-Probe-Quencher molecule, so the reporter dye can emit an increasedamount of fluorescence. Reporter dyes are not attached in any way to thesample. Free floating dyes can be floating freely in solution. Otherfluorescing markers well know in the art can be utilized. According tovarious embodiments, molecular beacons can be single-stranded moleculeswith hairpins that preferentially hybridize with an amplified target tounfold.

As shown in FIGS. 1, 5, 6 a, and 7, a detector 18 can be used to detectwhether an emission wavelength is emitted from the sample 12. Thedetector can be any suitable device known in the art, including, forexample, a photodiode, a charge coupled device (CCD), a camera, a CMOSdetector, or any combination thereof. The detector can be adapted torelay information to a data collection device for storage, correlation,and/or manipulation of data, for example, a computer, a signalprocessing system.

Multiple emission detectors 28, as shown, for example, in FIGS. 4 and 8,each set to receive a respective range of wavelengths of lightcorresponding to the emission wavelength of particular dyes, can also beused to detect any emitted light. According to various embodiments, themultiple emission detectors 28 can be arranged as an array. Varioustypes of multiple emission detectors for use according to variousembodiments are described, for example, in Publication No. WO 01/69211A1, which is incorporated herein in its entirety by reference.

According to various embodiments, the lens 14 can be a focusing lensthat can focus a wavelength of light emitted from a light source 16 intothe sample well 10. According to various embodiments, the lens 14 canfocus the emission wavelength light into a detector 18, as shown, forexample, in FIGS. 1, 6 a, and 7.

The lens can be any suitable lens configuration, including one or morelenses. The lens can be any lens known to practitioners in the art, forexample, a Fresnel lens or prism. A prism 26, as shown, for example, inFIG. 4, can be used in conjunction with a second lens 29. The prism 26can be arranged to direct the light of the excitation beam into thesample well 10, and the second lens 29 can be arranged to focus thelight of the emission beam into a detector or a detector array 28, asshown, for example, in FIG. 4. According to various embodiments, a prism26 can be used independently without any additional lenses. According tovarious embodiments, articles known in the art can be arranged to directthe light, for example, a prism, a grating, a mask, of the excitationbeam and/or emission beam.

According to various embodiments, the apparatus can include a lightsource, detector and a single lens, and can be substantially free ofother optical components, for example, mirrors, beamsplitters,additional focusing lenses, etc. According to various embodiments, theapparatus does not include a lens. According to various embodiments, thedistance between the light source and sample, and the distance betweenthe sample and detector can be proportional or thereabout to therelative distances illustrated in FIG. 5. According to variousembodiments, the light source can be spaced above the sample in therange from D/2 to 5D from the top surface of the sample, where D is thedepth of the sample in the well.

According to various embodiments, the light source 16 can include asource of light that can emit one or more respective wavelengths orwavelength ranges of light, for example, as shown in FIGS. 4 and 5, oran array 30 of two or more light sources 32, for example, as shown inFIGS. 1, 6 a, and 6 b.

According to various embodiments, a light array 30 includes two, three,four, or more sources of light 32 as shown, for example, in FIGS. 1, 6 aand 6 b. Each source of light 32 can emit a respective wavelength rangeof light to cause fluorescence of a different fluorescent dye. Thesources of light 32 can each include any suitable light emitting deviceor devices that emit a narrow wavelength of light, for example, a LED, alaser, a solid state laser, a laser diode, or any combination thereof.If a laser light source is used, it can be scanned in one or moredimensions. Super bright LEDs can be used because they are inexpensiveand can be arranged in a light array at low cost. According to variousembodiments, separate LEDs or a packaged set of LEDs can be used in anarray. Other suitable light sources will be apparent to practitioners inthe art. According to various embodiments, the light source can includea combination of two, three, or more LEDs, laser diodes, and the like,such as, for example, an LED emitting at about 475 nm, an LED emittingat about 539 nm, and a photodiode emitting at about 593 nm.

According to various embodiments, the light emitted from the lightsource 16, such as an LED or a laser, can be divided between twosamples, three samples, four samples, or more, reducing the number oflight sources needed per sample well array. An exemplary multiple samplewells layout is shown, for example, in FIG. 7. The light can be dividedbetween two or more sample wells 10 by any known device, including, forexample, one or more fiber optics, flood illumination device, one morelens with or without a mask, and/or one or more beam splitter.

According to various embodiments, a broad-spectrum light source, forexample, a white light or halogen light can be used as the light source16. According to various embodiments, the emitted light of thebroad-spectrum light source can be separated into distinct wavelengthsor wavelength ranges such that one wavelength or wavelength range at atime can be directed toward the sample 12. The light can be divided intodistinct wavelengths or wavelength ranges by an excitation wavelengthfocusing device, such as, for example, a prism (shown at 26 in FIG. 4),a diffraction grating (shown at 34 in FIG. 8), or a filter. Thedirection of a single wavelength or wavelength range can be controlledby use of an excitation wavelength-focusing device, such as, forexample, a diffraction grating or filter. According to variousembodiments, the broad spectrum light source can be filtered by way of alowpass filter, for example, to prevent light in the range of emissionwavelengths from reaching the sample. According to various embodiments,a pair of matching multi-notch filters can be used to prevent light inthe range of emission wavelengths from reaching the sample.

According to various embodiments, the apparatus can include anexcitation wavelength-excluding device 22, as shown in FIG. 1, forexample. The excitation wavelength-excluding device 22 can include, forexample, a diffraction grating, filter, prism, or mirror. The excitationwavelength-excluding device 22 can be positioned between the detector 18and the sample well 10. For example, according to various embodiments,the excitation wavelength-excluding device 22 can be positionedimmediately before and adjacent to a detector 18 as shown, for example,in FIGS. 1 and 7. According to various embodiments, the excitationwavelength-excluding device 22 can be a longpass filter which cantransmit wavelengths greater than about 600 nm to ensure thatsubstantially only the fluorescence emission wavelengths are received bythe detector or detector array. The excitation wavelength-excludingdevice can be positioned between a detector and a lens, or between alens and a sample as shown, for example, as a diffraction grating 34, inFIG. 8.

According to various embodiments, the excitation wavelength-excludingdevice can include multiple devices arranged in an array 36, as shown,for example, in FIG. 6 c. According to various embodiments, theexcitation wavelength-excluding device can be a prism 26 and can bepositioned between the multiple emission detectors 28 and the sample 12as shown, for example, in FIG. 4. According to various embodiments, theexcitation wavelength-excluding device can be a filter. Any filter knownin the art can be used, for example, a single bandpass or longpassfilter.

According to various embodiments, an excitation wavelength-focusingdevice can be arranged to ensure that substantially only excitationwavelengths, for example, wavelengths less than about 600 nm, areimpinged on the sample. The structure of these devices is as describedfor the excitation-wavelength excluding device. If the light source issufficiently monochromatic, the excitation wavelength-focusing devicecan be omitted. According to various embodiments, the excitationwavelength-focusing device can be located between the detector and thesample well, between a detector and a lens, or between a lens and asample well, for example.

According to various embodiments, a lightpipe 24 can be provided betweenthe light source and the sample, and/or between the detector and thesample, as shown, for example, in FIGS. 1 and 6 a. The lightpipe can belocated between the light source or detector and a lens, according tovarious embodiments. The lightpipe 24 allows the generation of anexcitation wavelength, and/or the detection of an emission wavelength,remotely from the sample well 10. The lightpipe 24 can be any suitabledevice for carrying wavelengths of light from one location to anotherwithout causing a significant loss of strength or shift in wavelength.According to various embodiments, such devices can include, for example,one or more fiber optic devices, mirrors, and/or lenses for transmittingthe light to the sample from a remote location, and/or for transmittingthe light from the sample to a remote location.

According to various embodiments, each individual light source can beprovided with a light delivering light pipe 40. Each light deliverylightpipe 40 can remain separate between the light source or detector,and the sample, or each light delivery lightpipe from the light sourceor detector can converge to form a single lightpipe at the sample end ofthe lightpipe, as shown, for example, in FIG. 6 a.

According to various embodiments, a method of differentiating multiplefluorescent signals by excitation wavelength can be provided. In themethod, a sample 12 including a plurality of components is provided in asample well 10. A plurality of fluorescent dyes is added to the sample12. The plurality of fluorescent dyes can be chosen such that theemission of each fluorescent dye is greatly increased when a particularcomponent is present in the sample 12. When the particular component ispresent in the sample 12, the dye can become unquenched.

Each of the plurality of detectable markers, for example, fluorescentdyes, can be selected to have an individual respective excitationwavelength or wavelength range. Each of the plurality of fluorescentdyes can be selected to possess an individual and respective emissionwavelength or wavelength range. The emission wavelength or wavelengthrange can overlap with an emission wavelength or wavelength range of oneor more of the other fluorescent dyes. A respective excitationwavelength or wavelength range is emitted from the light source 16 or30, toward the sample 12 to fluoresce a fluorescent dye, for example,when the dye is in the presence of a target analyte in the sample 12.Emission wavelengths and wavelength ranges that are emitted from thesample 12 within the emission wavelength range of the plurality offluorescent dyes, can be detected. The presence of a particularcomponent in the sample 12 can be determined by the detected emissionwavelength or wavelength range. The method can then be repeatedutilizing different excitation wavelengths emitted from the light source13 or 30 to determine the presence of other particular components in thesample 12.

According to various embodiments, the light source can be a LightEmitting Diode (LED). The LED can be, for example, an Organic LightEmitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), ora Quantum dot based inorganic “organic LED.” The LED can include aphosphorescent OLED (PHOLED).

As used herein, the terms “excitation source” and “light source” areused interchangeably.

According to various embodiments, excitation beams emitted from thelight source can diverge from the light source at an angle ofdivergence. The angle of divergence can be, for example, from about 5°to about 75° or more. The angle of divergence can be substantially wide,for example, greater than 45°, yet can be efficiently focused by use ofa lens, such as a focusing lens.

According to various embodiments, a light source can contain one LightEmitting Diode (LED) or an array of LEDs. According to variousembodiments, each LED can be a high power LED that can emit greater thanor equal to about 1 mW of excitation energy. In various embodiments, ahigh power LED can emit at least about 5 mW of excitation energy. Invarious embodiments wherein the LED or array of LEDs can emit, forexample, at least about 50 mW of excitation energy, a cooling devicesuch as, but not limited to, a heat sink or fan can be used with theLED. An array of high-powered LEDs can be used that draws, for example,about 10 watts of energy or less, about 10 watts of energy or more. Thetotal power draw can depend on the power of each LED and the number ofLEDs in the array. The use of an LED array can result in a significantreduction in power requirement over other light sources, such as, forexample, a 75 watt halogen light source or a 150 watt halogen lightsource. Exemplary LED array sources are available, for example, fromStocker Yale under the trade name LED AREALIGHTS. According to variousembodiments, LED light sources can use about 1 microwatt of power orless, for example, about 1 mW, about 5 mW, about 25 mW, about 50 mW,about 1 W, about 5 W, about 50 W, or about 100 W or more, individuallyor when in used in an array.

According to various embodiments, a quantum dot can be used as a sourcefor luminescence and as a fluorescent marker. The quantum dot based LEDcan be tuned to emit light in a tighter emission bandpass, thus thequantum dot based LED can increase the efficiency of the fluorescentsystem. Quantum dots can be molecular-scale optical beacons. The quantumdot nanocrystals can behave like molecular LEDs (light emitting diodes)by “lighting up” biological binding events with a broad palette ofapplied colors. Quantum dots can provide many more colors thanconventional fluorophores. Quantum dots can possess many other verydesirable optical properties. Nanocrystal quantum dots can be covalentlylinked to biomolecules using standard conjugation chemistry. The quantumdot conjugate can then be used to detect a binding partner in a widerange of assays. According to various embodiments, streptavidin can beattached to quantum dots to detect biotinylated molecules in a varietyof assays. Quantum dots can also be attached to antibodies andoligonucleotides. Any assay that currently uses, for example,fluorescent-tagged molecules, colorimetric enzymes, or colloidal gold,can be improved with quantum dot nanocrystal-tagged conjugates. Anexemplary quantum dot implementationis available from Quantum DotCorporation of Haywood, Calif. under the trademark QDOT. Moreinformation about quantum dots and their applications can be found at,for example, www.qdot.com. U.S. Pat. Nos. 6,207,229, 6,251,303,6,306,310, 6,319,426, 6,322,901, 6,326,144, 6,426,513, and 6,444,143 toBawendi et al., U.S. Pat. Nos. 5,990,479, 6,207,392, and 6,423,551 toWeiss et al., U.S. Pat. No. 6,468,808 to Nie et al., and U.S. Pat. No.6,274,323 to Bruchez et al., describe a variety of biologicalapplications, methods of quantum dot manufacturing, and apparatuses forquantum dot nanocrystals and conjugates, all of which are incorporatedherein by reference in their entireties.

Quantum dots can provide a versatile probe that can be used in, forexample, in multiplex assays. Fluorescent techniques using quantum dotnanocrystals can be much faster than conventional enzymatic andchemiluminescent techniques, can reduce instrument tie-up, and canimprove assay throughput. Colorimetric or detected reflectancetechniques can be inferior to fluorescence and difficulties ensue whenmultiplex assays are developed based on these materials. Quantum dotscan absorb all wavelengths “bluer” (i.e., shorter) than the emissionwavelength. This capability can simplify the instrumentation requiredfor multiplexed assays, since all different label colors can be excitedwith a single excitation source.

A Quantum dot based LED can emit light in an emission band that isnarrower than an emission band of a normal LED, for example, about 50%narrower or about 25% narrower. The Quantum dot based LED can also emitlight at an electrical energy conversion efficiency of about, 90% ormore, for example, approaching 100%. OLED films, including Quantum dotbased LEDs, can be applied to a thermal block, used for heating andcooling samples, in a fluorescence system without interfering with theoperation of the thermal block.

According to various embodiments, when an OLED is used, the OLED canhave any of a variety of sizes, shapes, wavelengths, or combinationsthereof. The OLED can provide luminescence over a large area, forexample, to luminescence multiple sample wells. Scatter or cross-talklight between multiple sample wells for this single OLED can be reducedby either overlaying a mask on the OLED or by patterning the luminescentin the OLED to operatively align with the multiple sample wells. TheOLED can be a low power consumption device. Examples of OLEDs in variousconfigurations and wavelengths are described in, for example, U.S. Pat.No. 6,331,438 B1, which is incorporated herein by reference in itsentirety. The OLED can include a small-molecule OLED and/or apolymer-based OLED also known as a Light-Emitting Polymer (LEP). Asmall-molecule OLED that is deposited on a substrate can be used. AnOLED that is deposited on a surface by vapor-deposition technique can beused. An OLED can be deposited on a surface by, for example,silk-screening. An LEP can be used that is deposited by, for example,solvent coating.

According to various embodiments, an OLED is used and can be formed fromone or more stable, organic materials. The OLED can include one or morecarbon-based thin films and the OLED can be capable of emitting light ofvarious colors when a voltage is applied across the one or morecarbon-based thin films.

According to various embodiments, the OLED can include a film that islocated between two electrodes. The electrodes can be, for example, atransparent anode, a metallic cathode, or combinations thereof. Severalseparate emission areas can be stimulated between a single set ofelectrodes where simultaneous illumination of the separate emissionareas is required. According to such embodiments, only one power andcontrol module might be required for several apparent light sources. TheOLED film can include one or more of a hole-injection layer, ahole-transport layer, an emissive layer, and an electron-transportlayer. The OLED can include a film that is about one micrometer inthickness, or less. When an appropriate voltage is applied to the film,the injected positive and negative charges can recombine in the emissivelayer to produce light by means of electroluminescence. The amount oflight emitted by the OLED can be related to the voltage applied throughthe electrodes to the thin film of the OLED. Various materials suitablefor fabrication of OLEDs are available, for example, from H.W. SandsCorp. of Jupiter, Fla. Various types of OLEDs are described, forexample, in U.S. Pat. No. 4,356,429 to Tang, U.S. Pat. No. 5,554,450 toShi et al., and U.S. Pat. No. 5,593,788 to Shi et al., all of which areincorporated herein by reference in their entireties.

According to various embodiments, an OLED can be used and produced on aflexible substrate, on an optically clear substrate, on a substrate ofan unusual shape, or on a combination thereof. Multiple OLEDs can becombined on a substrate, wherein the multiple OLEDs can emit light atdifferent wavelengths. Multiple OLEDs on a single substrate or multipleadjacent substrates can form an interlaced or a non-interlaced patternof light of various wavelengths. The pattern can correspond to, forexample, a sample reservoir arrangement. One or more OLEDs can form ashape surrounding, for example, a sample reservoir, a series of samplereservoirs, an array of a plurality of sample reservoirs, or a sampleflow path. The sample path can be, for example, a channel, a capillary,or a micro-capillary. One or more OLEDs can be formed to follow thesample flow path. One or more OLEDs can be formed in the shape of asubstrate or a portion of a substrate. For example, the OLED can becurved, circular, oval, rectangular, square, triangular, annular, or anyother geometrically regular shape. The OLED can be formed as anirregular geometric shape. The OLED can illuminate one or more samplereservoirs, for example, an OLED can illuminate one, two, three, four,or more sample reservoirs simultaneously, or in sequence. The OLED canbe designed, for example, to illuminate all the wells of a correspondingmulti-well array.

According to various embodiments, one or more excitation filters can beincorporated into the OLED substrate, thus eliminating additionalequipment and reducing the amount of space needed for an optical system.For example, one or more filters can be formed in a layer of a substrateincluding one or more OLEDs and a layer including a sample flow path.The wavelength emitted by the OLED can be tuned by printing afluorescent dye in the OLED substrate, as taught, for example, by Hebneret al. in “Local Tuning of Organic Light-Emitting Diode Color by DyeDroplet Application,” APPLIED PHYSICS LETTERS, Vol. 73, No. 13 (Sep. 28,1998), which is incorporated herein by reference in its entirety. Whenusing multiple emission lines in an OLED, the OLED can be used incombination with a multiple notch emission filter.

According to various embodiments, an OLED can be substituted in place ofany of the systems, devices, or apparatuses where an LED is shown. TheOLED light source can have several OLED films stacked and operativelydisposed such that several wavelengths of excitation beams can traversethe same optical path to illuminate the sample well. Several OLEDsforming excitation beams of the same wavelength can be stacked toprovide higher output to illuminate the sample well.

According to various embodiments, a sample well can be placed in betweenan excitation source and a detector. The sample well can be a microcard, for example, a microtiter card, such as a 96-well microtiter card.The excitation source can be, for example, an OLED, standard LED, orcombination thereof.

According to various embodiments, the light source can be a Solid StateLaser (SSL) or a micro-wire laser. The SSL can produce monochromatic,coherent, directional light and can provide a narrow wavelength ofexcitation energy. The SSL can use a lasing material that is distributedin a solid matrix, in contrast to other lasers that use a gas, dye, orsemiconductor for the lasing source material. Examples of solid statelasing materials and corresponding emission wavelengths can include, forexample: Ruby at about 694 nm; Nd:Yag at about 1064 nm; Nd:YVO4 at about1064 nm and/or about 1340 nm and which can be doubled to emit at about532 nm or about 670 nm; Alexandrite at from about 655 nm to about 815nm; and Ti:Sapphire at from about 840 nm to about 1100 nm. Micro-wirelasers are lasers where the wavelength of an excitation beam formed bythe laser can be tuned or adjusted by altering the size of a wire.According to various embodiments, other solid state lasers known tothose skilled in the art can also be used, for example, laser diodes.The appropriate lasing material can be selected based on the fluorescingdyes used, the excitation wavelength required, or both.

If a SSL is used, the laser can be selected to closely match theexcitation wavelength of a fluorescent dye. The operating temperature ofthe system can be considered in selecting an appropriate SSL. Theoperating temperature can be regulated or controlled to change theemitted wavelength of the SSL. The light source for the laser can be anysource as known to those skilled in the art, such as, for example, aflash lamp. Useful information about various solid state lasers can befound at, for example, www.repairfaq.org/sam/lasers1.htm. Examples ofsolid state lasers used in various systems for identification ofbiological materials can be found in, for example, U.S. Pat. No.5,863,502 to Southgate et al. and U.S. Pat. No. 6,529,275 B2 toAmirkhanian et al.; both of which are incorporated herein by referencein their entireties.

According to various embodiments, various types of light sources can beused singularly or in combination with other light sources. One or moreOLEDs can be used with, for example, one or more non-organic LEDs, oneor more solid state lasers, one or more halogen light sources, orcombinations thereof.

According to various embodiments, a light source can be used to provideexcitation beams to irradiate a sample solution containing one or moredyes. For example, two or more excitation beams having the same ordifferent wavelength emissions can be used such that each excitationbeam excites a different respective dye in the sample. The excitationbeam can be aimed from the light source directly at the sample, througha wall of a sample container containing the sample, or can be conveyedby various optical systems to the sample. An optical system can includeone or more of, for example, a mirror, a beam splitter, a fiber optic, alight guide, or combinations thereof.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with a light source to form the excitation beam. One or morefilters can be located between the one or more light sources and asample. One or more emission filters can be associated with an emissionbeam from an excited dye. One or more filters can be located between thesample and one or more emission beam detectors.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with one or more light sources to form at least oneexcitation beam. One or more filters can be located between the one ormore light sources and a sample. One or more emission filters can beassociated with an emission beam from an excited dye. One or morefilters can be located between the sample and one or more emission beamdetectors.

According to various embodiments, a filter can be a single bandpassfilter or a multiple bandpass filter. As used herein, a bandpass filterand a passband filter are used interchangeably. A multiple passbandfilter can be, for example, a multiple-notch filter or a multi-rugatefilter. A multiple passband filter can be used with an incoherent lightsource, for example, a halogen lamp, a white light source, and/or one ormore LEDs or OLEDs emitting light at different wavelengths. A multiplepassband filter can be used with a multiple laser-based light sourceemitting light at different wavelengths. Examples of manufacturing anduse of rugate filters and rugate beam splitters can be found in, forexample, U.S. Pat. No. 5,863,502 to Southwell, U.S. Pat. No. 6,256,148to Gasworth, and U.S. Pat. No. 6,529,275 B2 to Rahmlow, Jr., all ofwhich are incorporated herein by reference in their entireties.

According to various embodiments, a multiple passband filter can be usedwith a dichroic beam splitter, a 50/50 beam splitter, a dichroic beamsplitter that has several “passbands,” or no beam splitter. A multiplebeam splitter can be coated at an angle, causing a variance in athickness across a filter substrate, to compensate for wavelength shiftwith an angle. A multiple passband filter can be formed by coatingdifferent light interference materials over respective areas of asubstrate used in a multiple passband filter manufacture.

A Rugate filter is an example of an interference coating based on therefractive index that varies continuously in a direction, for example,perpendicular or 45 degrees to the film plane. When the refractive indexvaries periodically within two extreme values, a minus filter with hightransmittance on either side of the rejection band can be made. PeriodicRugate filters can be manufactured.

Rugate notch filters can use refractory metal oxides to achieve coatingswith exceptional thermal and environmental stability. These filters canbe used in place of other types of notch filters, particularly wheredurability and reliability are desired. Rugate notch filters areavailable from Ban Associates (Westford, Mass.). The Rugate notch filtercan be used as edge filters and beam splitters. Filter sizes or shapesare not limitations for the rugate notch filter. The rugate notch filtercan provide environmental and thermal stability, a broad operatingtemperature range, narrow rejection bands, variety of shapes & sizes,high throughput, low ripple, and/or a broad spectral range. Moreinformation is available from, for example, www.barr-associates-uk.com,www.barrassociates.com/opticalfilters.php.

Multiple-notch filters can be made, for example, with a measuredblocking of O.D. 6 or better. Notch filters with this type of deepblocking level at the light wavelength can also afford high transmissionclose to the light line.

According to various embodiments, excitation levels can increase whenmultiple dyes spaced apart spectrally are irradiated with excitationbeams. This can lead to less spectral crosstalk. The dye matrix,condition number, and/or deconvolution in a system can be improved. Theincreased excitation levels can provide higher signal levels. Highersignal levels can be seen during the utilization of dyes that emit inthe “red” spectrum. The dynamic range of the system can be improved. Thesystem can reduce the compensation for variation in the emission beamintensity for various dyes.

According to various embodiments, the multiple dyes can be deposited ina sample well using a fluid transfer technique, such as, for example,manual pipette, robotic pipette, or injection. According to variousembodiments, the multiple dyes can be deposited in a sample well, forexample, by ink-jet spraying, as beads, or as a mixture of a pluralityof dyes.

FIG. 9 is a bottom view that illustrates an OLED layout 400 that can beused as a light source, together with a plurality of photodiodedetectors 412, according to various embodiments. The OLED layout 400 caninclude a plurality of OLED well lamps 402, each positioned, when inoperation, above a respective well of a multi-well sample well array.Each OLED material well lamp 402 can be connected to, or integrallyformed with, a respective connection arm 404 that leads to a layoutterminal 406. Each layout terminal can be connected to or integrallyformed with the respective connection arms 404 branching from the layoutterminal.

The connection arms 404 branch off of side terminals 406 and 408. TheOLED layout can be connected to respective opposite electricalconnections, for example, opposite terminals of a power supply. The OLEDlayout can be connected to the power supply through leads arranged atopposite corners of the OLED layout. The power supply can include or beconnected to one or more of a switch, a meter, an oscillator, apotentiometer, a detector, a signal processing unit, or the like.Alternatively, or additionally, connection arms 404 can each include awire or electrical lead in the form of, for example, a metal wire. TheOLED layout can include a plurality of individually addressable OLEDlighting elements (not shown) with a separate lead connected to eachlighting element. The wiring, leads, terminals, connection arms, and thelike can be implemented in, for example, a substrate or a film. An OLEDlayout control unit 410 can be used to supply power and control the OLEDlayout 400. A plurality of detectors 412 can be electrically connectedto a detector control unit 416 through respective detector leads 414 asshown.

The plurality of detectors can be arranged, for example, centered, onthe plurality of OLED well lamps 402, on the sides of well lamps thatface respective sample wells, and/or when operatively positionedadjacent a multi-well sample well array. The detectors can be configuredto detect light emitted from the sample wells of a sample well array,without being flooded or bleached out by the respective OLED well lamps.For example, a mask material can be disposed between the detectors andthe respective OLED well lamps. The detector 412 can be formed in thesame substrate as the OLED lamp.

The exemplary OLED layout shown in FIG. 9 is shaped to be aligned with a24 well sample well array. Other embodiments of OLED layouts usingvarious shapes and various numbers of well lamps are within the scope ofthe present teachings.

According to various embodiments, each well lamp 402 can include, forexample, four individual lamps or OLED layers, capable of producingexcitation wavelengths at four different frequencies.

The OLED layout can be constructed of a unitary or multi-partconstruction, of molded material, of stamped material, of screen printedmaterial, of cut material, or the like.

FIG. 10 illustrates an exemplary embodiment of a light source layout. AnOLED layout 450 can include varying color OLEDs 452, 454, and 456stacked upon each other. The layout can be useful for a compact lightsource design capable of forming excitation beams at varyingwavelengths. The OLEDs 452, 454, and 456 can be transparent, allowingexcitation beams from each OLED to pass through any other OLED so as tobe directed towards a sample. The OLEDs 452, 454, and 456 can emitdifferent colors, same colors, or a combination thereof depending on thecolor intensity and variety required. The OLEDs 452, 454, and 456 canshare an electrode, for example, a cathode. One electrode, for example,an anode, for powering each of the OLEDs 452, 454, and 456 can beconnected in electrical isolation from each respective anode to acontrol unit (not shown) if the capability to independently activateeach of the OLEDs 452, 454, and 456 is desired. The OLEDs 452, 454, and456 can electrically share one electrode, two electrodes, or noelectrodes. Any number of OLEDs can be stacked, for example, two OLEDs,three OLEDs, four OLEDs, or more OLEDs, to form a light source, arespective light source, or an array of light sources.

According to various embodiments, as shown in FIGS. 12-14, one or moretransition filter 2061, 2062 such as, for example, a longpass filter, ashort pass filter, a beamsplitter, a prism, or a diffraction grating,can be located between the collimating lens 2020 and the plurality ofreaction regions 2040. According to various embodiments, the transitionfilter can be a long pass filter.

According to various embodiments, as shown in FIGS. 12-14, transitionfilters 2061, 2062 can pass bundles of collimated excitation beams 2025,2026 and reflect emission beams 2085 using the exemplary arrangementsshown including the LED source 2010 and detector 2080. According tovarious embodiments, transition filters 2061 and/or 2062 canindividually be positioned at a 45° angle or at angles other than 45°.Although transition filters 2061 and 2062 can split the optical paths ofthe bundles of collimated excitation beams 2025, 2026 from the emissionbeam 2085, other variations that achieve such a result are also suitableand can be used. For example, it can be desirable to minimize oreliminate the LED source light reaching detector 2080, and a dichroiclong pass filter used as transition filters 2061 and 2062 can be used toachieve this minimization. According to various embodiments, anon-dichroic long pass filter can be used for one or both of transitionfilters 2061 and 2062.

According to various embodiments, a transition filter can be positionedsuch that the filter is located along an excitation beam path between anLED source and a single reaction region. According to various otherembodiments, a transition filter 2061, 2062 can be located between oneor more LED source and two or more reaction regions, as shown, forexample, in FIGS. 12 and 13. According to various embodiments, thetransition filter 2061, 2062 can be located in an excitation beam pathbetween a collimating lens and a focusing lens, a reaction region lens,or a reaction region. According to various embodiments, the transitionfilter 2061, 2062 can be located in an emission beam path between areaction region, reaction region lens, or focusing lens, and a detector.

According to various embodiments, an LED source can emit excitationbeams towards multiple reaction regions in one row of reaction regions,or in two or more rows of reaction regions. For example, each LED sourcein FIG. 13 can illuminate reaction regions in one row. According tovarious embodiments, each LED can illuminate two reaction regions in twoor more adjacent rows.

According to various embodiments, the sample can contain a fluorescentdye or marker that fluoresces when un-quenched in the presence of atarget nucleic acid sequence to which the dye can bind. Fluorescent dyeprobes can be used. Other dyes that have similar characteristics can beused. The samples can also contain an additional, passive dye that canserve as a reference or control

If a reference dye is included, it can include, for example, of anucleic acid sequence labeled with a Rhodamine and/or Fluorescein dye orderivative thereof. An example of a suitable reference dye is ROX dyeavailable from Applied Biosystems of Foster City, Calif. The passive dyemolecule can be selected so as not to take part in a reaction, forexample, a PCR reaction, so that fluorescence from the passive dye issubstantially without influence from a target nucleic acid sequence andremains constant during the PCR. Fluorescence detected from the passivedye can be used to normalize the fluorescence from the target sequencebinding dye by using a standard concentration of the passive dye in oneor more of the reaction regions.

The LED source can emit excitation beams that include a secondaryexcitation frequency that causes the passive dye to fluoresce at asecondary emission frequency. The secondary emission frequency can bedirected to a detector to generate corresponding secondary data signals.The processor can receive the secondary data signals and computesecondary data representative of the known standard concentration of thepassive dye. These data can be used to normalize the primary data, sothat, for example, the concentration of the target nucleic acid sequenceis normalized to the standard concentration of the passive dye aftercorrecting the concentration computations of the target sequence inproportion to adjustments made in exposure time, and in conjunction withnormalization for drift, accounted for by analyzing the secondaryemission frequency. Greater details about the use of passive dyes andmathematical transformations using data collected from passive dyes areset forth in the ABI Prism 7000 Sequence Detection System User Guide,pages A-1 through A-10, available from Applied Biosystems, which isincorporated herein in its entirety by reference. The secondaryexcitation frequency can be identical to the primary excitationfrequency, and the passive dye can be selected to fluoresce such thatthe secondary emission frequency can be substantially at the emissionfrequency of the primary emission beams. In the example of PCR, theprimary data signals can be generated during each extension phase ofthermal cycling when the target sequence is recombined and the primarydye emission is maximized. The secondary data signals can be generatedduring each denature phase of thermal cycling when the target sequenceis denatured and correspondingly primary dye emission is minimized.Thus, data signals for the primary phase can be substantiallyrepresentative of the target sequence concentration, and data signalsfor the secondary phase can be substantially representative of thestandard concentration of passive dye.

Suitable excitation and emission filters for use in an opticalinstrument can be any conventional optical bandpass filters utilizing,for example, optical interference films, each having a bandpass at afrequency that is optimal for either the excitation wavelength of thefluorescent dye or the emission wavelength of the fluorescent dye. Eachfilter can have very high attenuation for non-bandpass frequencies toprevent “ghost” images from being reflected and to prevent stray light.For SYBR Green dye, for example, the excitation filter bandpass cancenter around a 485 nm wavelength, and the emission filter bandpass cancenter around a 555 nm wavelength. As shown in FIGS. 12-14, transitionfilters 2061, 2062 can transition from reflection to transmission at awavelength between these two, for example, at about a 510 nm wavelength,so that light of frequencies less than the transition wavelength can bereflected and higher wavelength light can pass through the filter, orvice versa. In this manner, according to various embodiments, atransition filter can function as one or more of an excitation filterand an emission filter.

According to various embodiments, multiple excitation wavelengths can beused to detect multiple sample components. According to variousembodiments, the apparatus and method can be adapted for use by anysuitable fluorescence detection system. For example, various embodimentsof the apparatus and method can be used in a sequencing system withsingle or multiple samples, for example, in a nucleic acid sequenceamplification reaction, in a sequencing detection system.

Various embodiments of the teachings are described herein. The teachingsare not limited to the specific embodiments described, but encompassequivalent features and methods as known to one of ordinary skill in theart. Other embodiments will be apparent to those skilled in the art fromconsideration of the present specification and practice of the teachingsdisclosed herein. It is intended that the present specification andexamples be considered as exemplary only.

1-33. (canceled)
 34. An apparatus comprising: a reaction region holdingassembly configured to hold a plurality of reaction regions andcomprising a thermal cycler block configured to affect a polymerasechain reaction; an excitation light source capable of providing at leastsix separate excitation beams, each of at least two of the excitationbeams including a wavelength range that is not provided by the other(s)of the at least two excitation beams, the excitation light source beingcapable of directing each of the at least six excitation beams along abeam path toward a single reaction region to which none of the otherexcitation beams are directed; a plurality of corresponding beamsplitters configured to receive respective ones of the plurality ofseparate excitation beams, each beam splitter disposed along an opticalpath between the excitation light source and a respective one of thereaction regions; a control unit capable of individually activating eachof the plurality of excitation beams independently from activating theother ones of the plurality of excitation beams; and a plurality ofdetectors, each detector capable of detecting emission beams emittedfrom a single reaction region from which none of the other detectors iscapable of detecting emission beams; wherein each beam splitter isdisposed along a respective emission beam path between a respective oneof the reaction regions and a respective one of the detectors.
 35. Theapparatus of claim 34, wherein the excitation light source comprises aplurality of excitation wavelength sources each capable of forming arespective excitation beam having a wavelength range, wherein eachexcitation beam wavelength range is exclusive of any other excitationbeam wavelength range.
 36. The apparatus of claim 34, wherein at leastone of the detectors comprises a photodiode, a charge coupled device(CCD), a camera, a CMOS detector, or any combination thereof.
 37. Theapparatus of claim 34, wherein the excitation light source comprisesseparate light emitting diodes.
 38. The apparatus of claim 34, whereinthe excitation light source comprises a light emitting diode emitting atabout 475 nm, a light emitting diode emitting at about 539 nm, and alight emitting diode emitting at about 593 nm.
 39. The apparatus ofclaim 34, further comprising a plurality of reaction regions held by thereaction region holding assembly, wherein the plurality of reactionregions comprises a plurality of sample wells.
 40. The apparatus ofclaim 34, wherein the excitation light source provides excitation beamsto irradiate a sample solution containing more than one dye.
 41. Theapparatus of claim 34, further comprising excitationwavelength-excluding devices, each excitation wavelength-excludingdevices being positioned between a detector and the reaction regionholding assembly.
 42. The apparatus of claim 34, wherein, duringoperation: the excitation light source provides a first excitation beamhaving a first wavelength that is directed to a first reaction regionheld by the reaction region holding assembly; the excitation lightsource provides a second excitation beam having a second wavelength thatis directed to a second reaction region held by the reaction regionholding assembly; the excitation light source is configured so that thefirst reaction region is illuminated by light from the excitation lightsource that is within the first wavelength range and is not illuminatedby light from the light source that is within the second wavelengthrange; and the excitation light source is configured so that the secondreaction region is illuminated by light from the excitation light sourcethat is within the second wavelength range and is not illuminated bylight from the light source that is within the first wavelength range.43. The apparatus of claim 34, wherein the plurality of detectorscomprises a combination of photodiodes.
 44. The apparatus of claim 34,wherein at least one of the beam splitters comprises a dichroic beamsplitter.
 45. An apparatus comprising: a plurality of reaction regionsconfigured to receive a plurality of respective samples and comprising athermal cycler block configured to affect a polymerase chain reaction;an excitation light source comprising at least six LEDs, each of atleast two of the LEDs characterized by a wavelength range that is notprovided by the other(s) of the at least two LEDs, each LED of the atleast six LEDs disposed above, and having optical access to, one of thereaction regions to which the other LEDs of the at least six LEDs do nothave simultaneous optical access; a set of at least two beam splitters,the set of at least two beam splitters disposed along a set of opticalpaths between the at least two LEDs and the plurality of reactionregions; a control unit capable of individually activating each of theat least six LEDs independently; and a plurality of detectors, eachdetector configured to detect an emission beam emitted from a respectivereaction region of the plurality of reaction regions when illuminated byan LED of the at least six LEDs; wherein the set of beam splitters isdisposed along a set of emission beam paths between the reaction regionsand the plurality of detectors.
 46. The apparatus of claim 45, whereinthe LEDs are configured so that during operation, the first reactionregion is illuminated by a color of the first LED and is not illuminatedby a color of the second LED, and the second reaction region isilluminated by the color of the second LED and is not illuminated by thecolor of the first LED.
 47. The apparatus of claim 45, wherein: theplurality of reaction regions comprises a first reaction region and asecond reaction region adjacent the first reaction regions; the at leastsix LEDs comprises a first LED disposed above the first reaction regionand a second LED disposed above the second reaction region; and thefirst LED and the second LED simultaneously illuminate the adjacentreaction regions.
 48. The apparatus of claim 45, wherein the at leastsix LEDs comprises a two-dimensional array of LEDs.
 49. The apparatus ofclaim 45, wherein, during operation, each LED is associated with onlyone reaction region such that at least one of the reaction regions isilluminated with light having a wavelength range that is notsimultaneously received by the other reaction region(s).
 50. Theapparatus of claim 45, wherein the plurality of detectors comprises acombination of photodiodes.
 51. The apparatus of claim 45, wherein atleast one of the beam splitters comprises a dichroic beam splitter. 52.An apparatus comprising: a reaction region holding assembly configuredto hold a plurality of reaction regions each containing a respectivesample; a thermal cycler block configured to affect a polymerase chainreaction on the samples; at least six modules, each module comprising aseparate light source, the at least six modules comprising a firstmodule and a second module; and a control unit capable of individuallyactivating the first light source independently from activating thesecond light source; wherein the first module is configured toilluminate a first reaction region of the plurality of reaction regions,the first module comprising: a first light source configured to providea first excitation beam comprising a first excitation wavelength rangedirected to the first reaction region; a first detector configured todetect an emission beam emitted from a sample located within the firstreaction region when the first reaction region is illuminated by thefirst excitation beam; and a first beam splitter configured to pass thefirst excitation beam and reflect the first emission beam; wherein thesecond module configured to illuminate a second reaction region of theplurality of reaction regions, the second module comprising: a secondlight source configured to provide a second excitation beam comprising asecond excitation wavelength range directed to the second reactionregion, the second wavelength range being different from the firstwavelength range; a second detector configured to detect an emissionbeam emitted from a sample located within the second reaction regionwhen the second reaction region is illuminated by the second excitationbeam; and a second beam splitter configured to pass the secondexcitation beam and reflect the second emission beam; wherein duringoperation, light at the first wavelength range is directed along a firstpath to the first reaction region and light at the second wavelengthrange is directed along a second path to the second reaction region, thefirst path and the second path containing no portions that overlap withone another; wherein the first excitation beam comprises a wavelengthrange that is not provided by the second excitation beam; and whereineach detector detects an emission beam emitted from a reaction regionthat is not detected by the other detector.
 53. The apparatus of claim52, wherein the first module comprises a first light delivery lightpipeand the second module comprises a second light delivery lightpipe,wherein the first lightpipe remains separate between the first lightsource and the first sample, and wherein the second lightpipe remainsseparate between the second light source and the second sample.
 54. Theapparatus of claim 52, wherein the first light source is disposed abovethe first reaction region and the second light source is disposed abovethe second reaction region.
 55. The apparatus of claim 52, wherein thelight sources are configured so that during operation, the firstreaction region is illuminated by light from the light sources that iswithin the first excitation wavelength range and is not illuminated bylight from the light sources that is within the second excitationwavelength range, and the second reaction region is illuminated by lightfrom the light sources that is within the second excitation wavelengthrange and is not illuminated by light from the light sources that iswithin the first excitation wavelength range.
 56. The apparatus of claim52, wherein the detectors comprise a combination of photodiodes.
 57. Theapparatus of claim 52, wherein at least one of the beam splitterscomprises a dichroic beam splitter.
 58. An apparatus comprising: firstand second reaction regions; a thermal cycler block configured to affecta polymerase chain reaction on samples contained within the reactionregions; at least six modules each comprising a separate light source,the at least six modules comprising a first module including a firstlight source and a second module including a second light source; and acontrol unit capable of individually activating the first light sourceindependently from activating the second light source; wherein the firstmodule comprises: the first light source consisting of a single firstLED characterized by a first wavelength range, the first light sourcedisposed above the first reaction region; a first detector configured todetect an emission beam emitted from a sample located within the firstreaction region when the first reaction region is illuminated by thefirst LED; and a first beam splitter configured to pass light within thefirst wavelength range and reflect the emission beam from the samplelocated within the first reaction region; wherein the second modulecomprises: the second light source consisting of a single second LEDcharacterized by a second wavelength range, the second light sourcedisposed above the second reaction region; a second detector configuredto detect an emission beam emitted from a sample located within thesecond reaction region when the second reaction region is illuminated bythe second LED; and a second beam splitter configured to pass lightwithin the second wavelength range and reflect the emission beam fromthe sample located within the second reaction region; wherein eachdetector is configured to detect an emission beam emitted from areaction region that is not detected by the other detector; and whereinthe wavelength range of the first light source is not provided by thewavelength range of the second LED light source.
 59. The apparatus ofclaim 58, wherein the first reaction region and the second reactionregion are adjacent to one another and the light sources are configuredto simultaneously illuminate the first reaction region and the secondreaction region.
 60. The apparatus of claim 58, wherein the single LEDsare configured so that during operation, the first reaction region isilluminated by LED light within the first wavelength range and is notilluminated by LED light within the second wavelength range, and thesecond reaction region is illuminated by LED light within the secondwavelength range and is not illuminated by LED light within the firstwavelength range.
 61. The apparatus of claim 58, wherein the first andsecond beam splitters are disposed along a common axis.
 62. Theapparatus of claim 58, wherein the detectors comprise a combination ofphotodiodes.
 63. The apparatus of claim 58, wherein at least one of thebeam splitters comprises a dichroic beam splitter.